Residual phosphorus and long-term management strategies for an Ultisol

Due to their extensive growth potential, transgenic root systems arising from inoculation withAgrobacterium rhizogenes became popular in the last decade as model systems in domains as diverse as production of secondary metabolites, interactions with pathogens and symbionts, examination of gene importance in control of root development or in regulation of gene expression in roots. Wild-type bacterial strains have also been considered as useful tools to stimulate rooting on recalcitrant cuttings or microcuttings as they cause abundant root initiation at the site of inoculation.Root initiation and the in vitro growth characteristics of transformed roots result from the transfer of genes located on the root-inducing plasmid (Ri) to plant cells and their expression therein. Two sets of pRi genes are involved in the root induction process: therol (rootloci) genes located in the TL region and theaux genes of the TR region. Some of these genes being able to interact, the system appears also as a new tool to study the role of auxin in the process of root initiation. The distinctive phenotype of the transformed roots which are capable of hormone autonomous growth seems to be controlled mainly by therol genes. Theserol genes, i.e. the geneticloci rol A, rol B, rol C androl D correspond to open reading frames ORFs 10, 11, 12 and 15. In vitro experiments determined the functions of the Rol B and Rol C proteins but the functions of Rol A and Rol D are still unknown. Altered metabolism of developmental regulators or modified sensitivity to auxin have been suspected to mediate root induction and morphological abnormalities of transformed roots and plants.The target cells for transformation and the cells which are competent for root initiation will be characterized as well as the subsequent development of transgenic roots provided with various constructs from the whole T-DNA to singlerol genes. Results dealing with auxin contents in relation with root growth kinetics, phenotype and structure, will also be presented and discussed with the potential use of therol genes to control root biomass. F J de Bruijn Section editor     

Independent induction of transformed roots by the TL and TR regions of the Ri plasmid of agropine type Agrobacterium rhizogenes

Summary To analyse the respective role of TL- and TR-DNA in root induction by agropine-type Agrobacterium rhizogenes Ri plasmids, deletions covering the TL- or the TR-regions were constructed in vitro and introduced into pRiA4 by marker exchange. Each T-region of pRiHRI was also cloned separately on an independent replicon and used in a binary system with the virulence functions of either an Ri or a Ti plasmid provided in trans. Transformed roots were induced on tobacco and tomato explants by TL-DNA as well as by TR-DNA, suggesting that agropine type Ri plasmids from strains A4 and HRI can induce root proliferation by two independent transformation mechanisms. The root induction by the TR-DNA is probably due to auxin biosynthesis by gene products of aux loci homologous to the tms genes of Ti plasmid T-DNA. The molecular mechanism of root proliferation induced by the TL-DNA is probably equivalent to that of mannopine type Ri plasmid T-DNA.     

Structure and function of root-inducing (Ri) plasmids and their relation to tumor-inducing (Ti) plasmids

The abilities of Agrobacterium tumefaciens and A. rhizogenes to transform dicotyle-dons and cause crown gall and hairy root disease are caused by the presence of tumor inducing (Ti) and root inducing (Ri) plasmids. During transformation plasmid T-DNA (transferred DNA) is inserted into the plant genome. The T-region is flanked by 25 bp direct repeats, which are essential for transfer. The T-regions contain oncogenes that are expressed in the plants. Some of these code for enzymes that synthesize auxin or cytokinin. Another type, present in Ri plasmids only, appears to impose a high hormone sensitivity on the infected tissue. The T-DNA also contains genes for enzymes synthesizing opines, which the bacteria catabolize. The T-DNA transfer is initiated by the induction of genes in the virulence (vir) region of the plasmid by phenolic compounds secreted by wounded tissue. The products of the vir -genes and of chromosomal genes mediate transfer of T-DNA to the plant cells. Crown gall disease is caused by production of auxin and cytokinin by the transferred T-DNA. The T-DNA of Ri plasmids codes for at least three genes that each can induce root formation, and that together cause hairy root formation from plant tissue. Current results indicate that the products of these genes induce a potential for increased auxin sensitivity that is expressed when the transformed cells are subjected to a certain level of auxin. After this stage the transformed roots can be grown in culture without exogenous supply of hormones.     

Identification of the genetic locus responsible for non-polar root induction by Agrobacterium rhizogenes 1855

Summary Root proliferation can be induced by Agrobacterium rhizogenes on carrot discs both on the apical and basal surface (facing the root apex and base, respectively) or on the apical surface only, depending on the bacterial strain. This differential response on the two surfaces is denominated polarity. We correlate the polarity of some strains with the absence of an Ri plasmid genetic locus, present in non polar strains such as A. rhizogenes 1855, which bears sequence homology with the auxin genes of Ti plasmid T-DNA. We demonstrate that this locus is responsible for root induction on the basal surface since insertion of a transposon in this region of pRi1855 induces polarity in this strain.     

Function of the aux and rol genes of the Ri plasmid in plant cell division in vitro

Auxin-autonomous growth in vitro may be related to the integration and expression of the aux and rol genes from the root-inducing (Ri) plasmid in plant cells infected by agropine-type Agrobacterium rhizogenes. To elucidate the functions of the aux and rol genes in plant cell division, plant cell lines transformed with the aux1 and aux2 genes or with the rolABCD genes were established using tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells. The introduction of the aux1 and aux2 genes enabled the auxin-autonomous growth of BY-2 cells, but the introduction of the rolABCD genes did not affect the auxin requirement of the BY-2 cells. The results clearly show that the aux genes are necessary for auxinautotrophic cell division, and that the rolABCD genes are irrelevant in auxin autotrophy.Key words: Agrobacterium rhizogenes, auxin-autotrophic cell, auxin biosynthesis, hairy root, plant cell division, Ri plasmid, T-DNA, aux, rol, tobacco BY-2 cells     

Further insight concerning the TL region of the Ri plasmid of Agrobacterium rhizogenes strain A4: Transfer of a 1.9 kb fragment is sufficient to induce transformed roots on tobacco leaf fragments

Summary Disarmed plant transformation vectors were used to assay the ability of subfragments of the T-regions of the Ri plasmid of agropine-type strain A4 of Agrobacterium rhizogenes to induce proliferation of transformed roots on tobacco leaf fragments. We have shown that a 6 kb region of TR-DNA, bearing the presumptive auxin synthesis genes, is capable of inducing transformed roots with an essentially normal phenotype as had been shown previously with the entire TR-region. A 1.9 kb fragment of the 20 kb TL-region is suffcient to induced transformed roots in the absence of exogenous hormones. These roots grow profusely on hormone-free medium, as is typical of roots transformed by the intact TL-DNA.     

A disarmed binary vector from Agrobacterium tumefaciens functions in Agrobacterium rhizogenes

Summary Binary Ti plasmid vector systems consist of two plasmids in Agrobacterium, where one plasmid contains the DNA that can be transferred to plant cells and the other contains the virulence (vir) genes which are necessary for the DNA transfer but are not themselves stably transferred. We have constructed two nononcogenic vectors (pARC4 and pARC8) based on the binary Ti plasmid system of Agrobacterium tumefaciens for plant transformation. Each vector contains the left and right termini sequences from pTiT37. These sequences, which determine the extent of DNA transferred to plant cells, flank unique restriction enzyme sites and a marker gene that functions in the plant (nopaline synthase in pARC4 or neomycin phosphotransferase in pARC8). After construction in vitro, the vectors can be conjugatively transferred from E. coli to any of several Agrobacterium strains containing vir genes. Using A. rhizogenes strain A4 containing the resident Ri plasmid plus a vector with the nopaline synthase marker, we found that up to 50% of the hairy roots resulting from the infection of alfalfa or tomato synthesized nopaline. Thus, vector DNA encoding an unselected marker was frequently co-transferred with Ri plasmid DNA to an alfalfa or a tomato cell. In contrast, the frequency of co-transfer to soybean cells was difficult to estimate because we encountered a high background of non-transformed roots using this species. Up to five copies of the vector DNA between the termini sequences were faithfully transferred and maintained in most cases suggesting that the termini sequences and the vir genes from the Ri and Ti plasmids are functionally equivalent.     

The aux1 gene of the Ri plasmid is sufficient to confer auxin autotrophy in tobacco BY-2 cells

Tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells are rapidly proliferating meristematic cells that require auxin for culture in vitro. We have established several transgenic BY-2 cell lines that carry the T-DNA of Agrobacterium rhizogenes 15834, which harbors an agropine-type root-inducing (Ri) plasmid. Two of these lines, BYHR-3 and BYHR-7, were used to test the role of auxin in the proliferation of plant cells. The lines grew rapidly in Linsmaier-Skoog (LS) medium lacking auxin and other phytohormones. The TR-DNA, containing the aux1 (tryptophan monooxygenase) and aux2 (indoleacetamide hydrolase) genes, was present in the genomes of both transgenic lines, whereas the TL-DNA, containing the rolA, B, C and D genes, was present in the genome of BYHR-7 but not BYHR-3. Since the introduction of the rolABCD genes alone did not affect the auxin requirement of BY-2 cells, the aux1 and aux2 genes, but not the rolABCD genes, appear to be relevant to the auxin autotrophy of these transgenic lines. Furthermore, the overexpression of aux1 allowed BY-2 cells to grow rapidly in the absence of auxin, suggesting the existence in plant cells of an unidentified gene whose product is functionally equivalent or similar to that of aux2 of the Ri plasmid.     

Regeneration of flax plants transformed by Agrobacterium rhizogenes

Regeneration of flax (Linum usitatissimum) following transformation by either Agrobacterium tumefaciens carrying a disarmed Ti-plasmid vector, or Agrobacterium rhizogenes carrying an unmodified Ri plasmid, was examined. Hypocotyl and cotyledon explants inoculated with A. tumefaciens formed transformed callus, but did not regenerate transformed shoots either directly or via callus. However, cotyledon explants inoculated with A. rhizogenes formed transformed roots which did regenerate transformed shoots. Ri T-DNA encoded opines were detected in the transformed plantlets and Southern hybridization analysis confirmed the presence of T-DNA from the Ri plasmid in their DNA. Transformed plantlets had curled leaves, short internodes and some had a more developed root system characterized by plagiotropic behaviour.     

Induction and growth properties of carrot roots with different complements of Agrobacterium rhizogenes T-DNA

Single and multiple infections of carrot discs were carried out with Agrobacterium strains harbouring different segments of pRi1855 TL-DNA cloned in the binary vector Bin 19 and with a strain carrying the TR-DNA from the same Ri plasmid. Roots induced by the various co-inoculations were cultured and their growth patterns were followed. Abundant roots could be induced by TL-DNA rol genes A, B and C as a single insert (rolA+B+C) and by rolB alone provided an extended segment beyond its 5 noncoding region was included in the construction. A depression of rooting capability was caused by the inclusion of rolC together with rolB (rolB+C). In all cases co-inoculation with the Agrobacterium carrying TR-DNA-borne auxin genes was necessary for root induction since none of the rol constructions was in itself capable of eliciting any response; an exceeding majority of these roots were however shown to contain rol genes but no TR-DNA. Rooting was also elicited if rol constructions were co-inoculated with a strain carrying TL-DNA genes 13 and 14 (ORF13+14) instead of the TR-DNA strain. These roots were shown to contain both rol genes and ORF13+14. Striking differences in growth properties were shown by roots containing different complements of TL-DNA genes. Typical hairy root traits, high growth rate, branching and, most noticeably, absence of geotropism, were shown by roots containing rolB alone, while roots with rolA+B+C were geotropic as normal carrot roots. Hairy root traits were conferred to rolA+B+C roots by the concomitant presence of ORF13+14 and by the addition of auxin to the culture medium. A model is presented which attempts to rationalize the growth patterns by assigning interplaying roles to the various TL-DNA genes involved.     

Callus,shoot and hairy root formation in vitro as affected by the sensitivity to auxin and ethylene in tomato mutants

We analyzed the impact of ethylene and auxin disturbances on callus, shoots and Agrobacterium rhizogenes-induced hairy root formation in tomato (Solanum lycopersicum L.). The auxin low-sensitivity dgt mutation showed little hairy root initiation, whereas the ethylene low-sensitivity Nr mutation did not differ from the control Micro-Tom cultivar. Micro-Tom and dgt hairy roots containing auxin sensitivity/biosynthesis rol and aux genes formed prominent callus onto media supplemented with cytokinin. Under the same conditions, Nr hairy roots did not form callus. Double mutants combining Rg1, a mutation conferring elevated shoot formation capacity, with either dgt or Nr produced explants that formed shoots with little callus proliferation. The presence of rol + aux genes in Rg1 hairy roots prevented shoot formation. Taken together, the results suggest that although ethylene does not affect hairy root induction, as auxin does, it may be necessary for auxin-induced callus formation in tomato. Moreover, excess auxin prevents shoot formation in Rg1.     

Multiple mutations in the transferred regions of the Agrobacterium rhizogenes root-inducing plasmids

Agrobacterium rhizogenes induces root formation at the site of inoculation in plants and inserts fragments of its Ri plasmid into the plant nuclear DNA. The transferred region (T-DNA) of the Ri plasmid of the A. rhizogenes strain A4 is made of two fragments, namely TL and TR; the latter harbors a sequence homology with the tms loci (responsible for auxin synthesis) of A. tumefaciens. On Daucus carota slices, single insertion mutations on the TL region of A. rhizogenes do not confer a mutant phenotype while an insertion-deletion in the TR region do confer a Basatt phenotype. Six double mutants with a single insertion in the TL region and the same deletion-insertion of the TR region were constructed. Three of these double mutants were avirulent on D. carota which indicates that in A. rhizogenes A4 the TL and the TR regions cooperate to confer a full infectious phenotype.     

Effects of light and growth regulators on adventitious bud formation in horseradish (Armoracia rusticana)

Summary To clarify that the presence of Ri T-DNA genes are not prerequisite for the light-induced bud formation in horseradish (Armoracia rusticana) hairy roots, leaf and root segments of nontransformed horseradish plants were used as explants. Bud formation from nontransformed tissues was observed in hormone-free medium under 16 h daylight conditions, but not under continuous darkness. To investigate the effects of growth regulators on bud formation, leaf and root explants were treated with auxin (1-naphthaleneacetic acid; NAA) and / or cytokinin (6-benzyl-aminopurine; BA). The most effective treatment in the dark to stimulate bud formation was BA at 1 mg·1^-1. These results show that adventitious bud formation in horseradish can be induced by light and growth regulators, and especially cytokinin, may be involved in bud formation, irrespective of whether the tissues were transformed with Ri T-DNA.Abbreviations BA 6-benzyl-aminopurine - NAA 1-Naphthaleneacetic acid - MS Murashige & Skoog (1962) medium     

发根农杆菌Ri质粒rol基因研究进展及在林木改良上的应用

Ri质粒上携带的诱根基因的表达不仅能导致植物被感染部位形成大量的毛根,而且由毛根容易获得再生的转化植株,这些植株可表现出许多能稳定遗传的表型变异,在植物遗传改良中有着广阔的应用前景。20世纪80年代以来,国内外学者对发根农杆菌Ri质粒及其rol基因进行了广泛深入的研究。本文重点综述了农杆碱型发根农杆菌Ri质粒的结构与功能,rol基因的位点与特征,Ri质粒的转化与rol基因的表达对植物生长发育的影响及在林木遗传改良上应用等方面的研究现状,并讨论了Ri质粒rol基因在林木遗传改良应用上存在的问题与应用前景。     

Binary plant vector based on Ri plasmid and part of T-DNA of the Ti plasmid

A binary vector system inA. tumefaciens for the introduction of foreign genes into the plant genome was developed. The first component is the Ri plasmid and the second component is a small vector plasmid, replicating inAgrobacterium, which carries the 25 bp terminal sequence of the Ti plasmid, theNos gene as the selectable marker and neighbouring sequences of the Ti plasmid. Functions necessary for integration are providedin trans by the virulence region of the Ri plasmid. Transformed cells are selected on the basis of hairy root tumor proliferation and agropine synthesis. They also showNos activity coded by the gene on the small part of Ti T-DNA.     

Leafy head formation of the progenies of transgenic plants of Chinese cabbage with exogenous auxin genes

The experiment was performed to evaluate the progenies of plant lines transgenic for auxin synthesis genes derived from Ri T-DNA.Four lines of the transgenic plants were self-crossed and the foreign auxin genes in plants of T5 generation were confirmed by Southern hybridization.Two lines,D1232 and D1653,showed earlier folding of expanding leaves than untransformed line and therefore had early initiation of leafy head.Leaf cuttings derived from plant of transgenic line D1653 produced more adventitious roots than the control whereas the cuttings from folding leaves had much more roots than rosette leaves at folding stage,and the cuttings from head leaves had more roots than rosette leaves at heading stage.It is demonstrated that early folding of transgenic leaf may be caused by the relatively higher concentration of auxin.These plant lines with auxin transgenes can be used for the study of hormonal regulation in differentiation and development of plant orgens and for the breeding of new variety with rapid growth trait.